Methylmercury (MeHg) is gradually changed to inorganic Hg after demethylation in animal tissues, and a selective quantification of inorganic Hg in the tissues is necessary to detect the reaction. We detected inorganic Hg formation in liver and kidney of mouse as early as 24 hr after MeHg injection. As an example of biological demethylation, the cytochrome P450 (P450)-mediated N-demethylation of drugs has been well documented, and formaldehyde was detected as a reaction product. Here we incubated mouse liver homogenate with added MeHg and observed a dose-dependent production of formaldehyde, as well as inorganic Hg formation. Since the amount of formaldehyde was approx. 500 times higher than that of the inorganic Hg that formed, the formaldehyde production would be stimulated by inorganic Hg formed from MeHg. We observed that inorganic Hg caused formaldehyde production, and it was enhanced by L-methionine and sarcosine. Thus, some biomolecules with S-methyl and N-methyl groups may function as methyl donors in the reaction. Using subcellular fractions of mouse liver, we observed that microsomal P450 did not participate in the demethylation of MeHg, but the greatest activity was located in the mitochondria-rich fraction. The addition of superoxide anion in the reaction mixture significantly enhanced the formaldehyde production, whereas Mn-superoxide dismutase depressed the reaction. Our present findings demonstrated that inorganic Hg formed by MeHg demethylation in mouse liver stimulated the endogenous formaldehyde production, and we observed that MeHg demethylation could be estimated by a formaldehyde analysis. Our results also suggested that superoxide anion is involved in the reaction.